Search

To investigate the potential immunomodulatory effects of concurrent ascariasis on the cytokine response to a live oral vaccine, we measured cytokine responses to cholera toxin B subunit (CT-B) following vaccination with the live oral cholera vaccine CVD 103-HgR in Ascaris lumbricoides-infected subjects randomized in a double-blind study to receive two doses of either albendazole or placebo prior to vaccination and in a group of healthy U.S. controls. Postvaccination cytokine responses to CT-B were characterized by transient increases in the production of interleukin-2 (IL-2; P = 0.02) and gamma interferon (IFN-gamma; P = 0.001) in the three study groups combined; however, postvaccination increases in IFN-gamma were significant only in the albendazole-treated A. lumbricoides infection group (P = 0.008). Postvaccination levels of IL-2 were significantly greater in the albendazole-treated group compared with the placebo group (P = 0.03). No changes in levels of Th1 and Th2 cytokines in response to control ascaris antigens were observed over the same period. These findings indicate that vaccination with CVD 103-HgR is associated with a Th1 cytokine response (IL-2 and IFN-gamma) to CT-B, that infection with A. lumbricoides diminishes the magnitude of this response, and that albendazole treatment prior to vaccination was able to partially reverse the deficit in IL-2. The potential modulation of the immune response to oral vaccines by geohelminth parasites has important implications for the design of vaccination campaigns in geohelminth-endemic areas.

Because concurrent infections with geohelminth parasites might impair the immune response to oral vaccines, we studied the vibriocidal antibody response to the oral cholera vaccine CVD 103-HgR in children infected with Ascaris lumbricoides and investigated the effect of albendazole pretreatment on the postvaccination response. Children with ascariasis were randomized to receive either 2 sequential doses of 400 mg of albendazole or placebo. After the second dose, CVD 103-HgR was given, and serum vibriocidal antibody levels were measured before and 10 days after vaccination. Postvaccination rates of seroconversion were greater in the treatment group that received albendazole (P=.06). Significantly greater rates of seroconversion and geometric mean titer were observed in the albendazole group in subjects with non-O ABO blood groups. A significant association was observed between vibriocidal seroconversion rates and treatment group, suggesting that A. lumbricoides infections impair the immune response to oral cholera vaccine, particularly in subjects of non-O blood groups.

A randomized, double-blind, placebo-controlled efficacy trial of one dose of CVD 103-HgR live oral cholera vaccine was performed in Indonesia from 1993 to 1997. 67,508 persons aged 2-41 years ingested vaccine or placebo and were followed for four years, detecting cholera cases using hospital-based surveillance. A nested reactogenicity study (538 vaccinees, 535 controls) revealed no vaccine-attributable side effects. A nested immunogenicity study (N=657) showed vibriocidal seroresponses in 64-70% of vaccinees vs 1-2% of controls. Cholera incidence was lower than expected. 103 cases of Vibrio cholerae O1 El Tor diarrhea were detected, 93 evaluable for vaccine efficacy (43 vaccine, 50 placebo; efficacy=14%). A suggestion of protection was observed among persons with blood group O [P=0.12]. Only seven cases occurred within six months of vaccination, precluding assessment of short-term efficacy. In Jakarta, single-dose CVD 103-HgR did not confer long-term protection. Short-term protection from a single-dose and long-term protection from two doses have yet to be studied.

CVD 103-HgR is a live oral cholera vaccine strain constructed by deleting 94% of the gene for the enzymatically active A subunit of cholera toxin from classical Inaba Vibrio cholerae O1 569B; the strain also contains a mercury resistance gene as an identifying marker. This vaccine was well tolerated and immunogenic in double-blind, controlled studies and was protective in open-label studies of volunteers challenged with V. cholerae O1. A randomized, double-blind, placebo-controlled, multicenter study of vaccine efficacy was designed to test longer-term protection of CVD 103-HgR against moderate and severe El Tor cholera in U.S. volunteers. A total of 85 volunteers (50 at the University of Maryland and 35 at Children's Hospital Medical Center/University of Cincinnati) were recruited for vaccination and challenge with wild-type V. cholerae El Tor Inaba. Volunteers were randomized in a double-blind manner to receive, with buffer, a single oral dose of either CVD 103-HgR (2 x 10(8) to 8 x 10(8) CFU) or placebo (killed E. coli K-12). About 3 months after immunization, 51 of these volunteers were orally challenged with 10(5) CFU of virulent V. cholerae O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Ninety-one percent of the vaccinees had a >/=4-fold rise in serum vibriocidal antibodies after vaccination. After challenge, 9 (39%) of the 23 placebo recipients and 1 (4%) of the 28 vaccinees had moderate or severe diarrhea (>/=3-liter diarrheal stool) (P < 0.01; protective efficacy, 91%). A total of 21 (91%) of 23 placebo recipients and 5 (18%) of 28 vaccinees had any diarrhea (P < 0.001; protective efficacy, 80%). Peak stool V. cholerae excretion among placebo recipients was 1.1 x 10(7) CFU/g and among vaccinees was 4.9 x 10(2) CFU/g (P < 0.001). This vaccine could therefore be a safe and effective tool to prevent cholera in travelers.

Several live oral vaccines (polio, bovine rotavirus, CVD 103-HgR cholera) are less immunogenic in developing than in industrialized countries. It was hypothesized that proximal small bowel bacterial overgrowth (common in children in less developed countries but rare in industrialized settings) diminishes the vibriocidal antibody response to CVD 103-HgR. In total, 202 fasting Santiago schoolchildren aged 5-9 years had lactulose breath H2 tests to detect proximal small bowel bacteria 1 day before ingesting CVD 103-HgR. Florid small bowel overgrowth was observed in 10 (5.6%) of 178 analyzable children. In children with florid overgrowth, vibriocidal seroconversion differed little from other children (60% vs. 67%), but the geometric mean titer was lower (160 vs. 368; P=.25). By logistic regression, increased peak breath H2 at small bowel time points was associated with diminished seroconversion (P=.04), as was the interaction of H2 value and weight (children >25 kg had lower seroconversion rates among subjects with heaviest overgrowth).

Background: Live oral cholera vaccine CVD 103-HgR is well-tolerated and immunogenic when administered to adults, school age children and preschool children in a single 5 x 10(9) colony-forming unit dose. Because elicitation of immune responses after administration of a single dose is exceptional for any oral vaccine in any age group, CVD 103-HgR was used as a probe to investigate the clinical acceptability, practicality and immunogenicity of this vaccine in infants and toddlers 3 to 17 months of age., Methods: The study was undertaken successively in 12- to 17-month-olds (n = 104), 7- to 11-month-olds (n = 106) and 3- to 5-month-olds (n = 102). One-half of the subjects were randomly allocated to receive vaccine and the other one-half to receive placebo, in double blind fashion. After 2 weeks of double blind follow-up, all subjects received a dose of vaccine. Vibriocidal antibody titers were measured on coded sera collected at baseline and 2 weeks after each dosing. The buffered vaccine "cocktail" had a volume of 100 ml; subjects who ingested > or =70 ml were considered fully vaccinated., Findings: Only 37% of subjects overall (25% of 3- to 5-month-olds) ingested > or =70 ml of the cocktail. The vaccine was well-tolerated with no significant differences in the rate or severity of adverse reactions after ingestion of vaccine vs. placebo. Seroconversion after ingestion of a single dose of CVD 103-HgR was similar in fully vaccinated subjects (66%) and in those who ingested a smaller fraction of the vaccine cocktail (63%). Of subjects who ingested two doses, 5 of 118 excreted vaccine organisms on Day 7 after the first dose vs. 0 of 118 after the second dose., Interpretation: Single dose oral CVD 103-HgR is well-tolerated and immunogenic in infants even when a partial dose is ingested. The buffered vaccine cocktail that is readily imbibed by older children is not appealing to young infants, and improved vaccine formulations and delivery vehicles for immunizing infants must be sought.

To provide optimum protection against classical and El Tor biotypes of Vibrio cholerae O1, a single-dose, oral cholera vaccine was developed by combining two live, attenuated vaccine strains, CVD 103-HgR (classical, Inaba) and CVD 111 (El Tor, Ogawa). The vaccines were formulated in a double-chamber sachet; one chamber contained lyophilized bacteria, and the other contained buffer. A total of 170 partially-immune American soldiers stationed in Panama received one of the following five formulations: (a) CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(7) CFU, (b) CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(6) CFU, (c) CVD 103-HgR alone at 10(8) CFU, (d) CVD 111 alone at 10(7) CFU, or (e) inactivated Escherichia coli placebo. Among those who received CVD 111 at the high or low dose either alone or in combination with CVD 103-HgR, 8 of 103 had diarrhea, defined as three or more liquid stools. None of the 32 volunteers who received CVD 103-HgR alone or the 35 placebo recipients had diarrhea. CVD 111 was detected in the stools of 46% of the 103 volunteers who received it. About 65% of all persons who received CVD 103-HgR either alone or in combination had a fourfold rise in Inaba vibriocidal titers. The postvaccination geometric mean titers were comparable among groups, ranging from 450 to 550. Ogawa vibriocidal titers were about twice as high in persons who received CVD 111 as in those who received CVD 103-HgR alone (600 versus 300). The addition of CVD 111 improved the overall seroconversion rate and doubled the serum Ogawa vibriocidal titers, suggesting that the combination of an El Tor and a classical cholera strain is desirable. While CVD 111 was previously found to be well tolerated in semiimmune Peruvians, the adverse effects observed in this study indicate that this strain requires further attenuation before it can be safely used in nonimmune populations.

Despite considerable experience with single-dose, live, oral cholera vaccine CVD 103-HgR in Asia, Europe, and the Americas, the vaccine had not been evaluated in sub-Saharan Africa or on individuals infected with human immunodeficiency virus (HIV). We therefore conducted a randomized, placebo-controlled, double-blind, cross-over clinical trial in 38 HIV-seropositive (without clinical acquired immunodeficiency syndrome (AIDS)) and 387 HIV-seronegative adults in Mali to assess its safety and immunogenicity. Adverse reactions (fever, diarrhoea and vomiting) were observed with similar frequency among vaccine and placebo recipients. The vaccine strain was not isolated from the coprocultures of any subject. The baseline geometric mean titre (GMT) of serum vibriocidal antibody was significantly lower in HIV-seropositives (1:23) than in HIV-seronegatives (1:65) (P = 0.002). Significant rises in vibriocidal antibody were observed in 71% of HIV-seronegatives and 58% of HIV-seropositives, and in 40% of HIV-seropositives with CD4+ counts below 500 per microliter. Following immunization, the peak vibriocidal GMT in HIV-seronegatives was 1:584 versus 1:124 in HIV-seropositives (P = 0.0006); in HIV-seropositives with CD4+ counts < 500 per microliter, the peak vibriocidal GMT was 1:40 (P = 0.03 versus other HIV-seropositives). CVD 103-HgR was safe in HIV-infected Malian adults, although serological responses were significantly attenuated among HIV-seropositives (particularly in those with CD4+ counts < 500 per microliter) relative to HIV-seronegatives. These results encourage further evaluations of this single-dose, oral cholera vaccine in high-risk populations such as refugees in sub-Saharan Africa.

To provide optimum protection against classical and El Tor biotypes of Vibrio cholerae O1, a single-dose, oral cholera vaccine was developed by combining two live, attenuated vaccine strains, CVD 103-HgR (classical, Inaba) and CVD 111 (El Tor, Ogawa). The vaccines were formulated in a double-chamber sachet; one chamber contained lyophilized bacteria, and the other contained buffer. In the first study, 23 U.S. adult volunteers received CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(8), 10(7), or 10(6) CFU, CVD 111 alone at 10(7) CFU, or placebo. In the second study, 275 Peruvian adults were randomized to receive CVD 103-HgR at 10(9) CFU plus CVD 111 at 10(9) or 10(8) CFU, CVD 111 alone at 10(9) CFU, CVD 103-HgR alone at 10(9) CFU, or placebo. Three of 15 U.S. volunteers who received CVD 111 at 10(7) or 10(8) CFU developed mild diarrhea, compared to none of 4 who received CVD 111 at 10(6) CFU and 1 of 4 who received placebo. Twelve (63%) of 19 vaccine recipients shed the El Tor vaccine strain. All but one volunteer developed significant Ogawa and Inaba vibriocidal antibody titers. Volunteers who received CVD 111 at 10(7) CFU had geometric mean Ogawa titers four to five times higher than those of volunteers who received the lower dose. In the second study, all dosage regimens were well tolerated in Peruvians. About 20% of volunteers who received CVD 111 at the high dose excreted the El Tor organism, compared to 7% in the low-dose group. CVD 111 was detected in the stools of two placebo recipients, neither of whom had symptoms or seroconverted. In all vaccine groups, 69 to 76% developed fourfold rises in Inaba vibriocidal antibodies. Among those who received the bivalent vaccine, 53 to 75% also developed significant rises in Ogawa vibriocidal antibodies. We conclude that it is feasible to produce a single-dose, oral bivalent vaccine that is safe and immunogenic against both biotypes (El Tor and classical) and both serotypes (Inaba and Ogawa) of cholera for populations in both developed and developing parts of the world.

A live oral cholera vaccine should ideally protect against both classical and El Tor biotypes of Vibrio cholerae O1. An El Tor biotype vaccine strain, therefore, would complement classical cholera vaccine strain CVD 103-HgR, a strain already in use in some countries. In this study, 25 healthy adult volunteers received a single dose of 10s colony-forming units of El Tor vaccine strain CVD 111, a derivative of El Tor Ogawa strain N16117 deleted in the virulence cassette. Three (12%) volunteers developed mild diarrhea (mean stool volume = 813 ml) but no systemic symptoms; 23 (92%) of the 25 volunteers developed serum vibriocidal antibodies (geometric mean titer = 1:2,291). Five weeks after vaccination, 18 vaccines and eight uninimunized control volunteers underwent wild-type challenge with El Tor Ogawa strain 3008. Three (16.7%) of 18 vaccinees and seven (87.5%) of eight controls developed diarrhea (P = 0.001) (vaccine efficacy = 80.9%). Further studies are underway to determine a dosage of CVD 111 that will be more clinically acceptable but equally immunogenic and protective.

Background: Cholera spread to Latin America in 1991; subsequently, cholera vaccination was considered as an interim intervention until long-term solutions involving improved water supplies and sanitation could be introduced. Three successive summer cholera outbreaks in northern Argentina and the licensing of the new single-dose oral cholera vaccine, CVD 103-HgR, raised questions of the cost and benefit of using this new vaccine., Methods: This study explored the potential benefits to the Argentine Ministry of Health of treatment costs averted, versus the costs of vaccination with CVD 103-HgR in the relatively confined population of northern Argentina affected by the cholera outbreaks. Water supplies and sanitation in this area are poor but a credible infrastructure for vaccine delivery exists., Results: In our cost-benefit model of a 3-year period (1992-1994) with an annual incidence of 2.5 case-patients per 1000 population and assumptions of vaccine efficacy of 75% and coverage of 75%, vaccination of targeted high risk groups would prevent 1265 cases., Conclusion: Assuming a cost of US$602 per treated case and of US$1.50 per dose of vaccine, the total discounted savings from use of vaccine in the targeted groups would be US$132,100. The projected savings would be altered less by vaccine coverage (range 75-90%) or efficacy (60-85%) changes than by disease incidence changes. Our analysis underestimated the true costs of cholera in Argentina because we included only medical expenditures; Indirect losses to trade and tourism had the greatest economic impact. However, vaccination with CVD 103-HgR was still cost-beneficial in the base case.

Although serum vibriocidal activity is used extensively as a marker of immunity to O1 Vibrio cholerae, there are limitations in this assay to detect instances of reexposure. We define the conditions operative in producing secondary vibriocidal responses in North American volunteers primed with either wild-type V. cholerae 1, 4, or 6 months later. Secondary serum vibriocidal responses occurred under two distinct secondary challenge conditions. The first occurred when secondary challenge produced a breakthrough in clinical protection. Following secondary exposure, 14 of 22 (64%) and 1 of 29 (3%) subjects with and without vibrio stool excretion, respectively, had secondary responses (P < 0.001); 5 of 6 (83%) and 10 of 45 (22%) subjects with or without diarrhea, respectively, mounted a secondary response (P = 0.006). The second condition occurred in the presence of full clinical protection but was dependent on the time interval between exposure. No subject (0 to 17) vaccinated with CVD 103-HgR and given homologous wild-type challenge within 4 months mounted a secondary vibriocidal response (P = 0.0009). The majority of the serum vibriocidal activity was of the immunoglobulin M (IgM) isotype, seen in 96 and 73% of subjects following primary and secondary exposure, respectively. Vibriocidal activity in the IgG fraction following primary and secondary exposures occurred with < or = 50% of volunteers; lipopolysaccharide (LPS)-specific IgG1 and IgG3 subclass responses supported the vibriocidal isotype data. However, following primary exposure, IgG4 LPS responses predominated, occurring in 81% of responding volunteers. These data suggest that, under certain conditions of secondary exposure to V. cholerae O1 antigens, when there is sufficient active local immunity present, there is a block of vibrio antigen resampling at the M cell level. We discuss the implications of and possible explanations for these findings.

Since October 1992, epidemics of cholera associated with Vibrio cholerae O group 139 have occurred in India, Bangladesh, and much of the rest of Asia. A volunteer model was used to determine the safety, immunogenicity, and efficacy of an attenuated delta ctxA delta zot delta ace delta cep V. cholerae O139 vaccine strain, designated CVD 112. Six volunteers received 10(6) cfu and 6 received 10(8) cfu of CVD 112. No subject who received the 10(6) dose had diarrhea or other severe symptoms after vaccination; 3 vaccinees developed mild diarrhea (mean stool volume, 648 mL) after receiving the higher dose. Five weeks after vaccination, 8 vaccinees and 15 unvaccinated control subjects underwent challenge with 10(6) cfu of wild type V. cholerae O139 AI1837. One vaccinee (13%) and 12 control subjects (80%) developed diarrhea after challenge (P = .003). The short-term protective efficacy conferred by vaccine strain CVD 112 was 84% and was remarkably similar to that conferred by primary wild type clinical infection (80%).

The safety and immunogenicity of the live oral attenuated vaccine strains vibrio cholerae CVD 103-HgR and Salmonella typhi Ty21a were evaluated alone or in a combined bivalent formulation in four groups composed of 185 healthy European adults. All presentations were well tolerated. The serum anti-S. typhi lipopolysaccharide immunoglobulin G and immunoglobulin A antibody responses were comparable for all groups (66 to 72% seroconversion). The serum vibriocidal antibody seroconversion rate ranged from 78 to 92.5% (P > 0.05) among the groups. However, the peak and geometric mean vibriocidal antibody titers were significantly higher (P < 0.005) in the groups which received the bivalent formulation along with two doses of Ty21a than in the group which received CVD 103-HgR followed by two doses of killed Escherichia coli K-12 placebo. The ingestion of a placebo shortly after CVD 103-HgR may have suppressed the magnitude of the immune response. These findings demonstrate the feasibility of producing multivalent live oral attenuated vaccines.

Persons of blood group O are at increased risk of developing cholera gravis. In a randomized, placebo-controlled, double-blind safety-immunogenicity trial of live oral cholera vaccine CVD 103-HgR in 5- to 9-year-old Chilean children, vibriocidal antibody seroconversion (74% overall) did not differ by blood group. However, the reciprocal geometric mean titer (GMT) in blood group O vaccines (GMT = 486) was higher than that in non-O vaccines (GMT = 179) (P < 0.02).

In many controlled studies, CVD103-HgR has been shown to be safe and immunogenic and to offer a significant degree of protection against experimental cholera after a single dose. Its minimal excretion and limited ability to compete and survive in various ecosystems indicate that this strain presents little risk to the environment. Furthermore, the potential of CVD103-HgR to regain virulence by acquisition of the CT A or LT A gene is extremely remote even under optimal conditions. Therefore, CVD103-HgR possesses those traits desired in a live oral attenuated vaccine produced by recombinant DNA technology.

The best correlate of protection against cholera is the level of serum vibriocidal antibodies, which are primarily directed against the O antigen of Vibrio cholerae O1 and lyse V. cholerae in the presence of complement. We established the timing of peak vibriocidal antibody response using sera from safety/immunogenicity studies of live oral cholera vaccines CVD 103-HgR, CVD 103-HgR2 and CVD 110 among immunologically naive North Americans and Colombians. The serum reciprocal vibriocidal antibody titre was consistently higher 10 days postimmunization than on either day 7 or day 14. This study suggests that recent phase 2 studies of CVD 103-HgR may have underestimated the peak vibriocidal titre by collecting serum on days 7-8 rather than on day 10; future studies of live oral cholera vaccines should take these results into account to obtain the best measurement of peak immunological responses. Because of the rapid drop in vibriocidal antibody titres about 2 weeks after immunization, care must be exercised in comparing immunogenicity of different vaccine candidates, formulations, dosage levels and immunization schedules.

The potential for reacquisition of ctxA genes by attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR was examined by performing a series of mating experiments under a variety of in vivo and in vitro conditions. We found no evidence that CVD 103-HgR could reacquire ctxA genes from wild-type V. cholerae O1 strains. However, if the donor V. cholerae O1 strains were genetically manipulated to add genes that allow chromosomal gene transfer, then ctxA sequences could be acquired by CVD 103-HgR. The minimal excretion of CVD 103-HgR by vaccinees and the refractoriness to reacquisition of ctxA sequences suggest that this well-tolerated, highly immunogenic live oral cholera vaccine will have a minimal environmental impact.

The current pandemic of cholera is caused primarily by Vibrio cholerae O1 of the El Tor biotype. Live attenuated classical biotype V. cholerae vaccine strains prevent severe and moderate cholera due to either biotype in challenged volunteers but may provide less protection against mild cholera due to El Tor organisms. CVD 110, a new ctxA-deleted vaccine strain derived from an El Tor Ogawa parent, lacks zona occludens toxin (Zot), accessory cholera enterotoxin (Ace), and hemolysin/enterotoxin. Ten healthy adult volunteers were given 10(8) cfu of CVD 110 with buffer; 7 developed diarrhea (mean stool volume, 861 mL). Vaccine organisms were shed in stool by all vaccines and were recovered from duodenal fluid in three-quarters of vaccinees. After vaccination, the geometric mean peak reciprocal vibriocidal titer among vaccinees was 17,829. CVD 110 is a powerful immunogen but insufficiently attenuated despite the absence of known potential enterotoxins of V. cholerae. Another unrecognized toxin or colonization alone may be responsible for diarrhea after ingestion of this strain.

The authors conducted a two-period crossover study of the reactogenicity and immunogenicity of live oral cholera vaccine CVD 103-HgR among US university students. Subjects ingested 5 x 10(8) colony forming units of either killed Escherichia coli K12 placebo or vaccine, followed by the opposite treatment one week later. Surprisingly, the dynamics of the immunologic response were influenced by prior ingestion of placebo. Subjects who received placebo first showed stronger vibriocidal antibody responses 2 weeks after vaccination compared with subjects who received vaccine first; this same pattern was seen for antitoxin titers. The authors suggest that ingestion of E. coli K12 one week prior to immunization boosts the immunologic response to vaccine by an unknown mechanism. Future crossover studies that examine immunologic outcomes might be designed to explore the ubiquity of such an effect.

Peripheral blood immunoglobulin A antibody-secreting-cell (ASC) responses are thought to reflect the mucosal immune response to locally presented antigens. We evaluated the ASC response to cholera toxin (CT) and Inaba lipopolysaccharide (LPS) in 26 North American volunteers following immunization with a single oral dose of live attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR and again upon homologous wild-type challenge with V. cholerae classical Inaba 569B. Challenge occurred at either 7, 30, or 180 days after vaccination. The CT and LPS ASC responses of volunteers following vaccination (83 and 55%, respectively) were similar in magnitude and frequency to those of unvaccinated controls following wild-type challenge (80 and 60%, respectively [0.1 < or = P < or = 0.9]). The responses were primarily immunoglobulin A. Vaccinated volunteers challenged within 30 days of vaccination had reduced or nondetectable CT and LPS ASC responses. Challenge at 6 months resulted in a heightened ASC response to LPS, confirming the existence of mucosal memory. ASC responses to CT upon challenge at 6 months were detectable but not different from that seen following primary immunization, suggesting that secondary ASC responses to different antigens from a single vaccine operate independently. In spite of these variable ASC responses, the vaccine efficacy was 100% following challenge for all vaccinees. V. cholerae-specific ASC responses following antigenic reexposure gave information on the presence of mucosal B memory cells but did not correlate with protective immunity. As such, these ASC assays will have limited usefulness for evaluating vaccine responders in vaccine field trials in cholera-endemic areas where prior V. cholerae O1 exposure is unknown.

One hundred years elapsed between the first (live, parenteral) cholera vaccine that entered clinical trials in 1885 and the field trials of two oral inactivated cholera vaccines undertaken in Bangladesh in the mid-1980s. The oral inactivated vaccines advanced the art by establishing, convincingly, that oral vaccines could protect (although multiple doses were required) and that (at least in adults) protection could last 3 years. Attenuated Vibrio cholerae O1 strain CVD 103-HgR (deleted of the cholera toxin A subunit gene and harbouring a gene encoding resistance to Hg++) constitutes another significant advance. This live oral vaccine is well tolerated and highly immunogenic in adults and children and highly protective (in adult volunteer challenge studies) following ingestion of of a single dose.

We conducted a double-blind, placebo-controlled, randomized crossover study to evaluate the safety and immunogenicity of a single 5 x 10(8)-CFU dose of live oral recombinant cholera vaccine CVD 103-HgR in 94 North American adults. The vaccine was well tolerated without associated adverse reactions. Despite minimal fecal excretion of vaccine, 97% of subjects exhibited serum vibriocidal antibody and 72% had antitoxin responses.

CVD 103-HgR is a live oral cholera vaccine that, in phase I and II studies to date, has been well tolerated and immunogenic. In challenge studies of US volunteers conducted 4-5 weeks after vaccination, CVD 103-HgR provided significant protection against experimental cholera due to classical and El Tor Vibrio cholerae O1. To determine the onset and duration of protection, two volunteer challenge studies were conducted: the first, 6 months after vaccination and the second, 8 days after vaccination. In both studies, CVD 103-HgR was 100% protective against diarrhea and significantly reduced the rate of shedding of vibrios after challenge with V. cholerae classical Inaba strain 569B, the virulent parent strain of CVD 103-HgR. Previously vaccinated subjects were less likely than naive controls to develop rises in titer of vibriocidal antibodies after challenge (P = .002), and the mean peak titer of vibriocidal antibodies was less than among controls. CVD 103-HgR can provide homologous protective immunity as soon as 8 days after vaccination and protection can persist for at least 6 months.

Adult volunteers received a booster dose (4 x 10(8) CFU) of attenuated Vibrio cholerae CVD 103-HgR oral vaccine 15 or 24 months after primary immunization. The immune response was modest, presumably due to rapid clearance of the vaccine strain by a primed immune system.

A randomized, double-blind, placebo controlled trial was conducted in 50 healthy Swiss adults to assess the safety and immunogenicity of the live oral attenuated cholera vaccine candidate strain Vibrio cholerae CVD 103-HgR (classical, Inaba). A single dose of 5 x 10(8) viable CVD 103-HgR organisms, administered in a buffered liquid formulation, was well tolerated as compared with individuals who received an equivalent amount of heat-killed Escherichia coli K-12 placebo. Eighty-eight percent of subjects receiving CVD 103-HgR mounted a significant (greater than fourfold) rise in Inaba vibriocidal titre while 68% did so for the heterologous Ogawa serotype. The magnitude of the vibriocidal antibody response (as measured by peak geometric mean titre and by fold-rise in titre over baseline) was greater for the homologous Inaba serotype. Nineteen out of 25 volunteers (76%) responded with a significant (p less than 0.05) rise in serum antitoxin levels. No vaccinee who received the E. coli K-12 placebo mounted a significant rise in either vibriocidal or antitoxin antibody levels. These results corrobrate the safety and immunogenicity of CVD 103-HgR in healthy adult volunteers.

Although great strides have been made in the development of recombinant attenuated Vibrio cholerae vaccine strains, the task has not been as simple as once imagined. The initial vaccine candidates proved to be unexpectedly reactogenic but further derivatives, such as CVD103-HgR, are well-tolerated, immunogenic and protective after a single dose. In addition, this strain carries a selectable marker to distinguish it from wild strains and has been evaluated in a practical, lyophilized formulation (Levine et al., 1988b). While CVD103-HgR is being further evaluated in expanded trials, we are also investigating a new secretogenic factor which could possibly explain the diarrhoea seen with the earlier vaccine strains. Hopefully, these studies will achieve the long-sought goal of a safe and effective vaccine for the prevention of cholera.

A live oral vaccine consisting of attenuated Salmonella typhi Ty21a expressing Vibrio cholerae O1 Inaba lipopolysaccharide (LPS) O antigen was constructed and tested in volunteers for safety, immunogenicity, and efficacy. Fourteen adults ingested three doses of 10(10) viable organisms with buffer. One month later, 8 vaccinees and 13 unimmunized controls were challenged with 10(6) pathogenic V. cholerae O1 E1 T or Inaba organisms. No significant adverse reactions to vaccination were observed. All volunteers had significant rises in serum immunoglobulin G (IgG) antibody to S. typhi LPS. Only 2 (14%) of 14 had significant rises in serum IgA or IgG antibody to Inaba LPS, and 5 (36%) of 14 had fourfold rises in vibriocidal antibody. In the challenge study, diarrhea occurred in 13 of 13 controls and 6 of 8 vaccinees (vaccine efficacy, 25%; P = 0.13). The vaccine significantly reduced the severity of the clinical illness (P less than 0.05) and caused decreased excretion of challenge vibrios (P less than 0.05). Although the typhoid-cholera hybrid vaccine did not provide significant protection overall against experimental cholera, this study demonstrates the importance of antibody to V. cholerae O antigen in ameliorating clinical illness and illustrates the use of an S. typhi carrier vaccine strain expressing a foreign antigen.

To address potential concerns over the release of genetically engineered live bacterial vaccines, we constructed a recombination-deficient derivative of the Vibrio cholerae O1 vaccine strain CVD103 (CVD103RM). Oral immunization of adult volunteers with CVD103RM showed that the recA mutation significantly diminished colonization ability and immunogenicity of the vaccine strain.

Natural protection from cholera is associated with local intestinal antibacterial and antitoxic antibodies, which appear to act synergistically. Although current parenteral cholera vaccines offer insufficient protection, new vaccines administered orally have more promise. Killed Vibrio cholerae, alone or given with the B subunit of cholera toxin, was evaluated in adult volunteers. Vaccinees, who received three doses of either vaccine, and unvaccinated controls ingested 10(6) V. cholerae organisms to determine the protective efficacy of the vaccines. The combination vaccine provided 64% protection, and the whole vibrio vaccine given alone provided 56% protection. In addition, illnesses in vaccines were milder than those in controls, and both vaccines gave complete protection against more severe disease. This substantial level of protection against a dose of V. cholerae that caused cholera in nearly 90% of controls suggests that these vaccines might provide at least as high a level of protection if given to the population of an endemic area. Indeed, a field efficacy trial is underway in Bangladesh, and preliminary data indicate a protective efficacy of 85% for a killed whole vibrio plus B subunit vaccine similar to that tested in volunteers and an efficacy of 58% for the killed whole vibrio vaccine alone. Thus, the studies in human volunteers were successful in predicting the substantial protection afforded by the vaccines in a cholera endemic area.

Texas Star-SR, a laboratory-derived mutant of Vibrio cholerae El Tor Ogawa 3083, which produces B but not A subunit of cholera toxin was given to 68 healthy adult volunteers in doses of 10(5) to 5 X 10(10) organisms. 16 of 68 exhibited loose stools but in only one individual was stool volume notable. Vomiting occurred in 1 and abdominal cramps in 3 vaccines; malaise and fever were not seen. Texas star was recovered from stools of 22% who received low doses (10(5) or 10(6) organisms) and from 63% who received high doses (10(8), 10(10), 2 X 10(10) or 5 X 10(10)). The attenuated strain was also recovered from jejunal fluid of 76% in the high dose group; cultures revealed 10(2)-10(5) organisms/ml. Seroconversions of vibriocidal antibody occurred in 93% and peak organisms/ml. Seroconversions of vibriocidal antibody occurred in 93% and peak titers were resembling those seen following clinical cholera. In contrast, serum IgG ELISA antitoxin rose significantly in only 29% and levels were below those encountered after clinical cholera. Only 5 of 18 vaccinees tested so far had significant rises in intestinal SIgA antitoxin; these also manifested rises in serum antitoxin. The occurrence of loose stools did not correlate with dose ingested, excretion of vibrios or rise in serum antitoxin. 503 clones recovered from coprocultures and jejunal fluids were negative when tested for enterotoxin. One month following a single 5 X 10(10) organism dose of Texas Star, 7 vaccinees and 6 controls were challenged with 10(6) pathogenic El Tor Ogawa vibrios. Diarrhea occurred in 7 of 10 controls but in only 1 of 7 vaccinees (p = 0.05). Despite clinical protection, excretion of pathogenic V. cholerae was similar in vaccinated and control groups. Twelve vaccinees who received two 10(9) or two 2 X 10(10) organisms doses of Texas Star 1 week apart were challenged with 10(6) pathogenic V. cholerae El Tor of the heterologous serotype. Diarrhea occurred in 11 of 15 controls but in only 3 of 12 vaccinees (p. 0.01). The 3 vaccinees with diarrhea all had mild illness. Texas Star-SR is a prototype that commonly causes mild diarrheal responses but is genetically stable in vivo and stimulates protective immunity against challenge with either the homologous or heterologous serotype.

An ideal vaccine does not yet exist to prevent cholera, a significant health problem in many less developed countries. Vibrio cholerae, the agent of epidemic and endemic cholera, colonizes the small bowel and secretes a potent enterotoxin that consists of a single A subunit, which stimulates adenylate cyclase activity, and five identical B subunits which bind to the ganglioside GM1 receptor of intestinal mucosal cells. Previous studies in man indicate that toxoid-derived antitoxic immunity by itself is insufficient to provide effective, long-lasting protection against cholera. Using recombinant DNA techniques we have now constructed a live, attenuated V. cholerae strain by deleting genes encoding the enterotoxin. Restriction enzyme fragments encoding cholera toxin were deleted in vitro from cloned vibrio chromosomal DNA and the resulting mutations introduced into the chromosome of a vibrio strain of proven immunogenicity. Recently, Mekalanos and coworkers have reported attenuated V. cholerae strains constructed by similar methods. It appears that recombinant DNA techniques offer a promising approach to the development of effective cholera vaccines.

The genes encoding the A (toxic) subunit of cholera toxin were deleted from pathogenic Vibrio cholerae O1 strain 569B by recombinant techniques, leaving intact production of immunogenic, non-toxic B subunit. The resultant strain, CVD 103, evaluated for safety, immunogenicity, and efficacy as a live oral vaccine, was highly attenuated and elicited strong antibacterial and antitoxic immune responses; a single dose significantly protected volunteers against challenge with pathogenic V cholerae O1 of either serotype or biotype. A further derivative, CVD 103-HgR, which has an Hg++-resistance gene to differentiate it from wild-type vibrios, was also well-tolerated, immunogenic, and protective; moreover, faecal excretion of this derivative was significantly lower than that of CVD 103, which should minimise environmental spread of the vaccine. CVD 103-HgR is a candidate for expanded clinical trials in endemic areas.

In recent years notable advances have been made in the development of improved vaccines to prevent cholera. These new vaccines are administered orally to maximally stimulate intestinal secretory immunity. Killed vibrios, given in conjunction with purified B subunit or administered alone, in three spaced doses, caused no adverse reactions and have conferred significant protection in volunteer challenge studies and in field trials. Two attenuated mutants of V. cholerae, prepared by recombinant DNA techniques, CVD 103 and CVD 103-HgR are well-tolerated and elicit prominent immune responses and protective immunity after ingestion of a single oral dose. Other modern approaches being pursued include the development of auxotrophic strains and of modifying attenuated S. typhi strain Ty21a to express V. cholerae Inaba and Ogawa LPS antigens.